NANO LETTERS
Telomerase-Generated Templates for the Growing of Metal Nanowires
2004 Vol. 4, No. 5 787-792
Yossi Weizmann, Fernando Patolsky, Inna Popov, and Itamar Willner* Institute of Chemistry and The Center for Nanoscience and Nanotechnology, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel Received January 11, 2004; Revised Manuscript Received February 19, 2004
ABSTRACT Telomers are single-stranded DNA consisting of constant repeat units that are generated by the ribonucleoprotein telomerase present in cancer cells (e.g., HeLa cancer cells). Two methods for using telomer templates for the preparation of Au nanowires are presented. By one method, telomerization is performed in the presence of aminoallyl-functionalized dUTP. The covalent tethering of an active ester-modified Au nanoparticle (1.4 nm) to the amine units followed by the catalytic enlargement of the particles yields the Au nanowires. The second method involves the hybridization of Au nanoparticles functionalized with a nucleic acid that is complementary to the telomere repeat units. The catalytic enlargement of the Au nanoparticles linked to the telomere duplex structure then yields the Au nanowire.
A recent challenge in nanotechnology is the preparation of metal nanowires that could act as circuitry components in future nanoscale devices.1 Several studies have addressed the use of biomaterials as templates for the assembly of metal nanowires and for the generation of “bottom-up” nanostructured materials. Silver wires were generated by selective deposition of the metal on self-assembled amyloid fibers.2 The use of DNA as a template for the generation of metal nanowires3 is of great interest for several reasons: (i) DNA templates of predesigned shapes, lengths, and base compositions can be synthesized. (ii) Different enzymes such as ligase, endonucleases, or polymerase provide catalytic tools for the elongation, scission, or replication of the desired templates. (iii) The binding of cations to the phosphate units or the intercalation of specific molecular compounds into double-stranded DNA provides the means to generate catalytic metallic seed clusters on the DNA or to link secondary components by the intercalating anchors. (iv) The base sequence of the nucleic acids encodes information in the DNA template, and thus, the specific metallization of specific sequences enables the fabrication of multimetal composite nanowires or patterned nanowires. (v) The sequence-specific binding of proteins to DNA allows the shielding of DNA domains while forming the metal nanowires. Significant advances in the synthesis of DNA-based nanowires have been reported in recent years. The reduction of the Ag+-phosphate site to Ag0 nanoclusters that catalyzed the formation of Ag nanowires,4 the deposition of different metals such as Pt,5 Pd,6 or Cu,7 and the patterning of proteinDNA complexes with Au nanowires,8 were reported. Also, * To whom all correspondence should be addressed. E-mail: willnea@ vms.huji.ac.il. Tel: 972-2-6585272. Fax: 972-2-6527715. 10.1021/nl049939z CCC: $27.50 Published on Web 04/03/2004
© 2004 American Chemical Society
the intercalation of psoralen-Au nanoparticles into DNA and the subsequent photochemical cross-linking of the intercalator to the DNA template were reported to yield nanoparticle wires.9 Recently, carbon nanotubes interconnected by Au nanowires generated on DNA templates were reported to be nanotransistor devices.10 However, the concept of preparing DNA-based nanowires suffers from several limitations. The synthetic preparation of DNA is limited by the length of the synthesized nucleic acid. However, natural DNAs usually lack the predesigned ordering of the base sequence of the DNA template. Furthermore, the construction of DNA-based metal nanocircuits for practical applications requires the highthroughput synthesis of the template and the resulting metal nanowire. Here we wish to report on the use of cancer cells as the catalytic reservoir for the preparation of long sequencecontrolled nucleic acid templates for the directed synthesis of metal nanowires. Telomerase is a ribonucleoprotein that synthesizes the chromosomal telomer ends (telomeric repeats) and is responsible for the continuous growth of cancer and malignant cells.11 In contrast to normal cells, where the erosion of telomers limits the cell replicative life span, in telomerasecontaining tumor cells the chromosomal telomer ends are not degraded, leading to indefinite cell proliferation of the cancer cells. Telomerase represents a special terminal reverse transcriptase with an estimated molecular mass of ∼1000 kDa. The active ribonucleoprotein is made up of an RNA strand and at least one catalytic protein component. The enzyme synthesizes telomeric repeats on the 3′ ends of human chromosomes using its integrated RNA as an internal template. Because telomerization introduces the constant sequence of the telomer repeats, the resulting nucleic acid
Scheme 1. Assembly of Au Nanowires on a Telomer Template via the Hybridization of a Au-Nanoparticle-Labeled Nucleic Acid Complementary to the Telomer and the Catalytic Enlargement of the Particles
template is encoded with specific, repeatable “base information” that could be used for the directed secondary synthesis of the metal wires. Furthermore, the telomerization process could generate, by a high-throughput process, long-chain templates. Scheme 1 depicts one method for the telomerase-induced synthesis of Au nanowires. The Au-nanoparticle-functionalized nucleic acid (oligo-Au) complementary to the telomeric repeats sequence was prepared by reacting the amino oligonucleotide (1) with mono-NHS Au nanoparticles of 1.4nm diameter (Nanoprobes Inc.). The telomer templates are prepared by the telomerization of the primer (2) in the presence of cancer cell extracts (HeLa cells). The resulting telomers are then hybridized with the complementary oligoAu hybrid. The resulting nanoparticle-decorated doublestranded DNA is then reacted with HAuCl4 and hydroxylamine (NH2OH) to enlarge the Au nanoparticles and to yield a continuous gold nanowire.12 Figure 1A shows a typical telomer strand that is formed upon telomerization of the primer by the telomerase-containing cell extract. The height of the DNA is ca. 0.8 nm, consistent with a single-stranded nucleic acid. Variable lengths of the synthesized telomers are observed, and telomers that reach lengths of 3.5 µm were detected. Hybridization of the telomers with 1 leads to nanoparticle-labeled double-stranded DNA. Figure 1B shows an example of nanoparticle-labeled DNA. It should be noted that although the wire structure seems to be continuous because of the tip dimensions, the particles are probably separated. The height of the Au-nanoparticle-labeled DNA is ca. 2 nm, consistent with the nanoparticle and DNA dimensions. Parts C and D of Figure 1 show AFM and TEM images of the metallic structures, respectively, obtained upon the enlargement of the Au-nanoparticle-labeled DNA using 788
Scheme 2. Assembly of Au Nanowires on a Polyamino-Functionalized Telomer Template by the Covalent Attachment of Active-Ester-Functionalized Au Nanoparticles to the Template, and Catalytic Enlargement of the Tethered Au Particles
HAuCl4 and NH2OH. Metallic structures with a height ranging from 10 to 40 nm are observed, consistent with the dimensions expected from the enlargement of the nanoparticle seeds. In this context, it should be noted that recent research efforts demonstrated the use of Au-nanoparticlefunctionalized nucleic acid as catalytic labels for the amplified detection of DNA.12,13 In these studies, the amplified microgravimetric detection of DNA using quartz crystal microbalance experiments12 and the analysis of DNA by increasing the conductivity between microelectrodes13 bridged by the enlarged particles were reported. The present study images the enlargement of the Au nanoparticles on the DNA template. The second approach to using telomerase originating from HeLa cancer cells as the biocatalyst for the generation of DNA templates for metallic nanowires is depicted in Scheme 2. The primer (2) is telomerized in solution in the presence of the dNTP mixture that includes dATP, dGTP, dTTP, and amino-dUTP (3). The resulting amino-labeled telomers are then reacted with the N-hydroxysuccinimide-functionalized Au nanoparticles to yield nanoparticle-labeled single-stranded DNA. The nanoparticles are subsequently used as catalysts for their enlargement with HAuCl4 and NH2OH to generate continuous metallic structures. In contrast to the previous method that employed double-stranded DNA as the template for the synthesis of the metallic nanowires, the present method uses single-stranded DNA as the template. The telomerization of the primer (2) was performed in an aqueous buffer solution using the enzyme telomerase, Nano Lett., Vol. 4, No. 5, 2004
Figure 1. (A) AFM image of the telomer template generated upon the telomerization of the primer (2) in the presence of the HeLa cell extract. (B and C) AFM images of the Au-nanoparticle wires generated according to Scheme 1 before and after the catalytic enlargement of the particles, respectively. (D) TEM image of the Au nanowire generated after the enlargement of the primary 1.4-nm particles hybridized with the telomer according to Scheme 1.
extracted from 50 000 HeLa cells, as a biocatalyst. The ratio between dTTP and amino-dUTP (dTTP/amino-dUTP) was 1:10, and it was optimized by primary PCR experiments followed by electrophoretic separation of the products and the determination of the extent of polymerization. We find that in the presence of dATP, dGTP, and amino-dUTP (3) but in the absence of unlabeled dTTP, the telomerase-induced enlongation of the primer (2) is completely blocked. Presumably, the enzyme is prohibited from sequentially introducing two labeled amino-dUTP nucleotides. The optimized ratio of dTTP/amino-dUTP leads to the formation of DNA templates with molecular lengths of ca. 1000 nm. At higher contents of 3, the telomers are substantially shorter. The covalent attachment of the Au nanoparticles to the amine Nano Lett., Vol. 4, No. 5, 2004
units of the telomer templates was performed in solution, and the resulting labeled DNA was deposited on the mica surfaces. Figure 2A shows a typical image of the resulting surface-confined structures. Surprisingly, all of the Aunanoparticle-labeled telomers form circles upon their deposition on mica. The diameter of most of the circles is in the range of 50 to 100 nm, which probably corresponds to cyclized single Au-labeled telomer chains. About 5% of the circles are substantially larger (ca. 400 nm), and the circle contours are deformed. We think that these structures are composed of several Au-labeled telomer templates that are coincidentally formed during the deposition process. The origin of the cyclization of the Au-nanoparticle-labeled telomers is not well understood. In a previous study,9 Au789
Figure 2. (A) AFM image corresponding to the Au-nanoparticle tethered telomers, according to Scheme 2, prior to their catalytic enlargement. (Bottom) Enlargement of the surface-confined nanostructures and their cross-sectional analysis. (B and C) AFM images of the Au nanowires prepared according to Scheme 2 after the catalytic enlargement of the particles. (D) TEM image of the catalytically enlarged Au wire generated according to Scheme 2. (Inset) HRTEM of a nanocluster generated in the Au wire.
nanoparticle-functionalized poly-L-lysine was reported to form nanoparticle circles on mica surfaces. The formation of the circles was attributed to the drying of aqueous nanodroplets on the surface. Because we observe the formation of such circles only with the Au-functionalized amino-modified telomers or the Au-nanoparticle-modified 790
poly-L-lysine, it might well be that the drying of the aqueous droplets on the surface yields contours of tightly bound water to the amine functions that lead to the geometrical circle contours. The height of the circles is ca. 2 nm (Figure 2A), consistent with the formation of a nanoparticle-modified DNA template. It should be noted that the amino-functionNano Lett., Vol. 4, No. 5, 2004
alized telomers (lacking the Au nanoclusters) do not form any circular structures on the mica. Thus, the circles are specific to the Au-nanoparticle-functionalized telomers. Enlargement of the Au-nanoparticle telomers with HAuCl4 and NH2OH in solution results in the formation of Au metallic nanowires. Parts B and C of Figure 2 depict two typical AFM images of metallic wires. We observe long metallic wires in the range of 1-3 µm, with a height that corresponds to ca. 10 nm. Figure 2D shows the TEM image of the metallic structure obtained upon the enlargement of the Au-nanoparticle-labeled DNA using NH2OH and HAuCl4. Interestingly, the enlargement of the particles leads to the formation of single-crystal-enlarged particles. The HRTEM (Figure 2D, inset) reveals that the wire is composed of gold crystallites with a diameter in the range of 50-80 nm. The crystals grow with the characteristic fcc equilibrium morphology. The inset in the figure shows the lattice image of the (111) faces of one of the crystallites. Fourier analysis did not reveal any perturbation in the interatomic distances (within the accuracy of TEM). It should be noted that the TEM images of the resulting nanowires (Figures 1D and 2D) do not always show direct contact between the enlarged particles, and gaps up to 15 nm between the particles are observed. The methods are, however, not optimized, and the improvement of the covalent attachment of the Au nanoclusters to the amino-functionalized telomers as well as the optimization of the time-interval for the enlargement process could lead to continuous interconnected metallic nanowires. We also observed in the TEM images that the enlarged nanoparticle metallic wires exhibit different shapes upon their preparation by the two methods. Whereas the wire generated by the hybridized Au nanoparticles reveal helical structures after enlargement and the attached particles turn along the helical template (Figure 1D), the enlarged Au-nanoparticle wire on the Au-nanocluster-linked amino-functionalized telomers reveals a flexible wire conformation. This is consistent with the flexibility of the latter single-stranded template. To conclude, we have introduced a novel approach for generating metallic nanowires on DNA templates. This method is based on the generation of DNA templates by telomerase originating from cancer cells. The method allows the generation of a constant sequence repeat along the template, thus enabling its sequential labeling with Au nanoparticles or the hybridization of Au-nanoparticle-labeled complementary oligonucleotides to the telomeric repeats. A major advantage of this method is the efficient synthesis of template units by telomerase originating from cancer cells. Furthermore, the recently reported telomerase-induced generation of telomers on nanoparticles14 (e.g., CdSe/ZnS coreshell nanoparticles) suggests that the telomerization of primers associated with nano-objects (e.g., semiconductor nanoparticles on carbon nanotubes and the subsequent metallization of the templates to generate electrical contacts) may lead to a versatile means of fabricating nanodevices such as nanotransistors. Experimental Section. Materials. All primers were purchased from Sigma Genosys Inc. and used as received. Nano Lett., Vol. 4, No. 5, 2004
Amino-dUTP, dATP, dGTP, dTTP, NH2OH, HAuCl4, and all other compounds were purchased from Sigma and used as received without further purification. Mono-N-hydroxysuccinimide-functionalized nanogold (1.4-nm diameter) was purchased from NanoProbes Inc. Telomerization Conditions. The telomerization reaction without labeling was performed in the presence of the primer (2, 10 µM), dGTP, dATP, and dTTP (0.2 mM each), and telomerase solution (20 mM Tris buffer, pH 8.3, 1.5 mM MgCl2, 0.63 mM KCL, 0.05% Tween 20, 1 mM EGTA) at 30 °C for 2 h. The telomerization reaction according to Scheme 2 was performed under the same conditions but in the presence of amino-dUTP and dTTP in a ratio of 10:1, respectively. All of the telomer templates resulting from the telomerization reactions were purified and isolated by the use of an UltraClean PCR Clean-up DNA purification kit (MoBio Laboratories, Inc.). Preparation of Oligonucleotide (1)-Nanogold Hybrids. A solution of amino-labeled oligo 1 (10 nmoles in Hepes buffer at pH 7.6, 10 mM) was reacted overnight with 6 nmoles of mono-N-hydroxysuccinimide-functionalized nanogold at room temperature. The oligonucleotide 1-nanoparticle hybrid was purified from free oligonucleotides by a centricon filtration device (10 000 cutoff, Millipore Inc.). Au nanoparticles were catalyticaly enlarged in the presence of 0.5 mM NH2OH and 0.5 mM HAuCl4 at room temperature for 20 s. All AFM samples were prepared on freshly cleaved mica surfaces by dropping 4 µL of the DNA sample solutions, followed by drying the sample in a clean environment. Acknowledgment. This research is supported by the German Israel Foundation (GIF). References (1) (a) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128-4158. (b) Katz, E.; Shipway, A. N.; Willner, I. In Nanoparticles: From Theory to Applications; Schmid, G., Ed., Wiley-VCH: Weinheim, Germany, 2004; Chapter 6, pp 368-421. (2) (a) Reches, M.; Gazit, E. Science 2003, 300, 625-637. (b) Scheibel, T.; Parthasarathy, R.; Sawicki, G.; Lin X. M.; Jaeger, H.; Lindquisat, S. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4527-4532. (3) Richter, J.; Mertig, M.; Pompe, W.; Vinzelberg, H. Appl. Phys. A 2002, 74, 725-728. (4) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yoseph, G. Nature 1998, 391, 775-778. (5) Richter, J.; Mertig, M.; Pompe, W.; Monch, I.; Schackert, H. K. Appl. Phys. Lett. 2001, 78, 526-538. (6) Mertig, M.; Ciacchi, L. C.; Seidel, R.; Pompe, W.; De Vita, A. Nano Lett. 2002, 2, 841-844. (7) Monson, C. F.; Woolley, A. T. Nano Lett. 2003, 3, 359-363. (8) Keren, K.; Krueger, M.; Gilad, R.; Ben-Yoseph, G.; Sivan, U.; Braun, E. Science 2002, 297, 72-75. (9) Patolsky, F.; Weizmann, Y.; Lioubashevski, O.; Willner I. Angew. Chem., Int. Ed. 2002, 41, 2323-2327. (10) Keren, K.; Berman, R. S.; Buchstab, E.; Sivan, U.; Braun, E. Science 2003, 302, 1380-1382. (11) (a) Moyzis, R. K.; Buckingham, J. M.; Cram, L. S.; Dani, M.; Deaven, L. L.; Jones, M. D.; Meyne, J.; Ratliff, R. L.; Wu, J. R. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 6622-6626. (b) Harley, C. B.; Futcher, A. B.; Greider, C. W. Nature 1990, 345, 458-460. (c) Wright, W. E.; Piatyszek, M. A.; Rainey, W. E.; Byrd, W.; Shay, J. W. ReV. Genet. 1996, 18, 173-179. (d) Shay, J. W.; Wright, W. E. Curr. Opin. Oncol. 1996, 8, 66-71. (e) Shay, J. W.; Bacchetti, S. Eur. J. Cancer 1997, 33, 787-791. (12) Weizmann, Y.; Patolsky, F.; Willner, I. Analyst 2001, 126, 15021504. 791
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